Magnesium (Mg) is the second most abundant cation in the body [Altura, B. M. et al., Drugs 28 (Suppl.I):120-142, 1984]. It is cofactor for more than 325 cellular enzymes involved in cellular energy production and storage, protein synthesis, DNA and RNA synthesis, cell growth and reproduction, adenylate cyclase synthesis, maintenance of cellular electrolyte composition, and stabilization of mitochondrial membranes [Altura, B. M. et al, Drugs 28 (Suppl.I):120-142, 1984; Wacker, W. E. C. Magnesium and Man, Harvard Univ. Press, Cambridge, 1980]. As a consequence of these biochemical activities, Mg plays a pivotal role in control of neuronal activity, cardiac excitability, neuromuscular transmission, muscular contraction, and vasomotor tone [Altura, B. M. et al., Drugs 28 (Suppl.I):120-142, 1984; Wacker, W. E. C. Magnesium and Man, Harvard Univ. Press, Cambridge, 1980; Altura, B. M. et al., in: Metal Ions in Biological Systems, ed. by H. Sigel and A. Sigel, Vol 26: Compendium on Magnesium and Its Role in Biology, Nutrition, and Physiology, pp 359-416, Marcel Dekker, Inc. New York, 1990].
Most clinical data of Mg determinations are derived from blood levels of total Mg (Wacker, W. E. C. Magnesium and Man, 1980; Elin, R. J. Clin. Chem. 33:1965-1970, 1987). Total serum Mg concentrations reflect protein-bound (30-40%), chelated (7-12%), and free or ionized Mg (Mg.sup.2+) (60-70%) fractions. The exact proportion of these fractions has been extremely difficult to determine precisely, and, moreover, there is no way to rapidly make such determinations. Precise information about Mg activity is pivotal to our understanding of Mg metabolism. The free or ionized form (Mg.sup.2+) is the active form of the mineral (Wacker, W. E. C. Magnesium and Man, 1980; Elin, R. J. Clin. Chem. 33:1965-1970, 1987; Ryan, M. F. Ann. Clin. Biochem. 28:19-26, 1991). Alterations in circulating protein levels (primarily albumin), which are seen in numerous pathophysiologic states, will alter the interpretation of Mg status (very similar to calcium) (Elin, R. J. Clin. Chem. 33:1965-1970, 1987).
Although numerous methods are available clinically, to determine total Mg in serum, plasma, urine, cerebral spinal fluid and other body fluids (e.g., atomic absorption spectrophotometry, atomic emission spectrophotometry, colorimetry, fluorometry, compleximetry and chromatograph for quantifying total Mg), none of these can determine ionized or free Mg.sup.2+ (Elin, R. J. Cin. Chem. 33:1965-1970, 1987; Wills, M. R. et al. Magnesium 5:317-327, 1986).
Until the present invention, the only method for assessing free Mg.sup.2+ in biological samples was an ultrafiltration procedure (Wacker, W. E. C. Magnesium and Man, 1980; Elin, R. J. Clin. Chem. 33:1965-1970, 1987; Wills, M. R. et al. Magnesium 5:317-327, 1986; Aikawa, J. K. Magnesium: Its Biologic Significance, CRC Press, Boca Raton, 1981). While this procedure is capable of measuring free Mg.sup.2+, it is fraught with a multiplicity of problems (need to control pH, need to control filter composition, time-consuming, inability to access whole blood Mg.sup.2+, need for centrifugation of blood). In addition, and most important, these classical methods, which primarily depend upon modifications of the procedure outlined by Watchorn, E. et al. (Biochem. J. 26:54, 1932), Toribara et. al. (J. Clin. Invest. 36:738, 1957) and Walser, M. (J. Clin. Invest. 40:723-730, 1961) result in ionized Mg.sup.2+ values on normal subjects which are significantly different from those obtained by the present method as assessed using an ion selective electrode (ISE). Using ultracentrifugation methods combined with ultrafiltration methods to assess free Mg.sup.2+, the percentages of ultrafilterable Mg reported by previous workers (around 70%) (Cummings, N. A. et al. Anal. Biochem 22:108-116, 1968; Nielson, S. P. Scand. J. Clin. Lab. Invest. 23:219-225, 1960) are much higher than the values using the present method. Even more recent measurements, using ultrafiltration and a micropartition filtration system has yielded a much wider range of values for ultrafilterable Mg from normal human subjects than those of the present method (D'Costa, M. et al. Clin. Chem. 29:519, 1983; Zaloga, G. P. et al. Crit. Care Med. 15:813-816, 1987). Some of these pitfalls preclude determination of Mg.sup.2+ in various body fluids. Moreover, determinations can not be done on less than 1.0 ml of blood.
The physiologic or pathophysiologic effects of mild to severe (or graded) decreases or increases in extracellular free Mg.sup.2+ in whole blood, serum or plasma has not been possible to discern in human subjects or animals either rapidly (e.g., within 1-2 min) or repeatedly (multiple samples over a few minutes-hours). Since Mg is frequently used in normomagnesemic patients for its antiarrhythmic, vasomotor and neuronal actions [Altura, B. M. et al. Drugs 28(Suppl.I):120-142, 1984; Wacker, W. E. C. Magnesium and Man, 1980; Altura, B. M. et.al. In: Metal Ions in Biological Systems, 1990; Iseri C. T. et al. West J. Med. 138:823-828, 1983; Ebel, H. et al. J. Clin. Chem. Clin. Biochem. 21:249-265, 1983], it is vital to be able to assess the exact extracellular level of ionized Mg.sup.2+ at any one instant. Although there is a dire need to carefully monitor extracellular Mg.sup.2+ in hypomagnesemic patients or patients linked to Mg deficiency states such as cardiovascular insufficiency, cardiac arrhythmias, coronary artery spasm, those at risk for sudden death, renal disorders, respiratory muscle weakness, pre-eclampsia, eclampsia, migraine, hypertension, premenstrual syndrome, tetany, seizures, tremor, apathy, depression, hypokalemia and hypocalcemia, there is at present no way to do this either precisely or rapidly [Altura, B. M. et al. Drugs 28(Suppl.I):120-142, 1984; Wacker, W. E. C. Magnesium and Man, 1980; Altura, B. M. et.al. In: Metal Ions in Biological Systems, 1990; Iseri, C. T. West J. Med. 138:823-828, 1983; Ebel, H. et al. J. Clin. Chem. Clin. Biochem. 21:249-265, 1983;Altura, B. M. et al. Magnesium 4:226-244, 1985; Zaloga, G. P. Chest 56:257-258, 1989; Sjogren, A. J. Intern. Med. 226:213-222, 1989; Zaloga, G. P. et al. In: Problems in Critical Care, ed. G. P. Zaloga Vol 4:425-436, J.B. Lippincott Co., Philadelphia, 1990; Resnick, L. M. et al. Proc. Nat. Acad. Sci. U.S.A. 81:6511-6515, 1984; Rudnick, M. et al. APMIS 98:1123-1127, 1990].
In 1980, it was suggested on the basis of in-vitro experiments that drops in ionized serum Mg.sup.2+ would produce coronary vasospasm, arrhythmias and sudden death (Turlapaty and Altura, Science 208:198-200, 1980). Although clinical observations from other workers in the intervening years have suggested this might be a "real" possibility, up until the present invention, no evidence could be gathered due to the unavailability of a method for accurate and rapid assessment of blood ionized Mg.sup.2+ (Altura, B. M. et al. In: Metal Ions in Biological Systems, Vol 26, 1990; Ebel, H. et al. J. Clin. Chem. Clin. Biochem. 21:249-265, 1983; Altura, B. M. et al. Magnesium 4:226-244, 1985; Sjogren, A. et al. J. Intern. Med. 226:213-222, 1989; Zaloga, G. P. et al. In: Problems In Critical Care Vol 4, 1990).
Over the past 10 years, it has been determined that reductions in ionized Mg.sup.2+, experimentally in animals and isolated cerebral blood vessels, can induce intense vasospasm and rupture of blood vessels in the brain (Altura, B. M. et al. In: Metal Ions in Biological Systems Vol 26, 1990; Altura, B. T. et al.Neuroscience Letters 20:323-327, 1980; Altura, B. T. et al. Magnesium 1:277-291, 1982; Altura, B. T. et al. Magnesium 3:195-211, 1984; Altura, B. M. et al. Am. J. Emerg. Med. 1:180-193, 1983; Huang, Q-F., et al. FASEB J. 3:A845, 1989). On the basis of such experimental findings, it has been hypothesized that head trauma would be associated with deficits in serum, plasma and whole blood ionized Mg.sup.2+ (Altura, B. T. et al. Magnesium 1:277-291, 1982; Altura, B. T. et al. Magnesium 3:195-211, 1984; Altura, B. M. et al. Am. J. Emerg. Med. 1:180-193, 1983). The present inventions has allowed these studies to be undertaken for the first time.
In the 1970's and 1980's, on the basis of numerous animal experiments, it was reported that deficits in ionized Mg.sup.2+ would result in maintained peripheral vasospasm, constriction of small blood vessels in numerous organ regions and as a consequence development of high blood pressure or hypertension (Altura, B. M. et al. Drugs 28 (Suppl.I): 120-142, 1984; Altura, B. M. et al. In: Metal Ions in Biological Systems Vol 26, 1990; Altura, B. M. et al. Magnesium 4:226-244, 1985; Sjogren, A. et al. J. Intern. Med. 226:213-222, 1989; Turlapaty, P. D. M. V. et al. Science 208:198-200, 1980; Altura, B. M. et al. Federation Proc. 40:2672-2679, 1981; Altura, B. M. et al., Science 221:376-378, 1983; Altura, B. M. et al. Science 223:1315-1317, 1984). Until the development of the present invention, this hypothesis was not testable because of a lack of proper methodology for processing samples and measuring ionized Mg.sup.2+.
Accelerated atherosclerotic heart disease is a leading cause of death in the long-term (&gt;10 year) renal transplant recipient. Hypertension and hyperlipidemia are common in this population and may be secondary to cyclosporine use. Cyclosporine has been associated with a renal tubular total magnesium (Twg) leak, as evidence by low serum total magnesium values and increased urinary excretion. Hypomagnesemia has been implicated as a factor in modulation of blood lipid levels, alteration of vascular tone and cyclosporine toxicity. Until the present invention, accurate measurements of biologically active ionized magnesium or ionized Ca.sup.2+ /ionized Mg.sup.2+ ratios were not possible. Therefore, until the present invention, it was not possible to determine the ratio of ionized calcium and ionized magnesium in hypercholesterolemia and cyclosporine toxicity in renal transplant recipients.
In 1981-1983, studies on isolated blood vessels from animals and pregnant women, suggested that reduction in dietary intake of Mg or inability to metabolize Mg properly could result in reduction in ionized Mg.sup.2+ and thus in umbilical and placental vasospasm, possibly reducing oxygen and nutrients to the growing fetus (Altura, B. M. et al. Federation Proc. 40:2672-2679, 1981; Altura, B. M. et al., Science 221:376-378, 1983). The end result could be, in large measure, responsible for fetal growth retardation, pre-eclampsia, hypertension and convulsions, particularly in pregnant indigent women (Rudnick, M. et al. APMIS 98:1123-1127, 1990; Altura, B. M. Science 221:376-378, 1983). Mg has been recommended as early as 1925 in this country for treatment and prevention of pregnancy-induced pre-eclampsia, hypertension and convulsions, but a method for accurately monitoring ionized Mg.sup.2+ rapidly and repeatedly throughout pregnancy was not available until development of the present invention.
National population statistics indicate that 18 million women and 5.6 million men suffer from severe migraine headaches..sup.1 Only about one-third of all headache patients are fully satisfied with treatments currently available.
Although major advances have been made in unraveling the etiologies of numerous disease states, the precise cause(s) of migraine headaches remains elusive. Many investigators suspect that migraine and tension-type headaches may be part-and-parcel of a similar biologic process despite having differing clinical symptoms..sup.2-4 One popular theory is that although a susceptible individual may first demonstrate intermittent migraines, these will evolve into chronic daily headaches, similar in nature to tension-type headaches, suggesting that possibly the two types of headaches derive from a similar initiating biochemical etiology. Another leading theory is that all headaches share a common neurotransmitter dysfunctional mechanism, for example, related to the actions of serotonin. Changes in magnesium levels have effects on the functions of serotonin,.sup.27-29 and NMDA receptors,.sup.30-32 nitric oxide production,.sup.33 catecholamine production and activity.sup.24 and on more than 325 enzymatic processes in the body..sup.35 IMg.sup.2+ levels are known to affect entry of Ca.sup.2+, intracellular Ca.sup.2+ levels and its release from sarcoplasmic and endoplasmic reticulum membranes in vascular muscle and endothelial cells which in turn control vascular tone and vascular reactivity to endogenous hormones and neurotransmitters..sup.36, 37 Such vascular effects could play a role in migraine pathogenesis..sup.5 Thus, magnesium deficiency could be involved in both the vascular and neurological aspects (e.g., migraine aura, pain, nausea, etc.) of the development of migraines.
Since 60 percent of all migraine sufferers report that close relatives often have a similar problem, it would appear that genetic control mechanisms are also important in several types of headaches. It is thus possible that, biochemically, there may be a common factor to the above enigma. A reliable biologic marker(s) for headache has long-remained elusive..sup.4
A large body of circumstantial evidence points to the possible role of magnesium deficiency in the pathogenesis of headaches and a possible vascular mechanism has been proposed..sup.5 It was pointed out there that all drugs which have been successfully utilized for the treatment or prevention of migraine attacks acts on cerebral blood vessels. Cerebral blood vessels are more sensitive to Mg (either, deficient or excess of) than any other type of vascular muscle cells; Mg deficiency resulting in contraction and potentiation of vasoconstrictors and excess resulting in vasodilatation and inhibition of vasoconstrictors..sup.5, 6, 38 Deficiency in Mg.sup.2+ in neuronal and vascular tissue could be expected to result in elevation in levels of catecholamines, serotonin, prostaglandins and substance P,.sup.34, 35, 38-41 vasoactive substances long implicated in the etiology of migraine..sup.5 In addition, serotonin is known to be released form platelets during a migraine attack,.sup.42 and to be a potent cerebral vasoconstrictorl and promote nausea and vomiting..sup.43 Some prostanoids, e.g., PGE.sub.1, are known to provoke pulsating headaches and additional migraine-like symptoms.sup.44 as well as potentiate vasoconstrictor hormones acting on blood vessels..sup.45 Pain in headache is often attributed to the action of substance P on sensory fibers..sup.46 It is of some interest to note that an Mg-deficiency induced state has recently been shown experimentally to result in significant generation and release of substance P into the blood stream..sup.41 Excess Mg is known to attenuate and inhibit the production and actions of substance P, serotonin and catecholamines on several types of end-organs including cerebral vascular tissue..sup.5,32,33,36,38
Since 1960,.sup.47 it has been known that Mg.sup.2+ can prevent spreading depression in the cerebral cortex. This spreading depression causes an attenuation of blood flow to the affected area of the brain, resulting in local hypoxia and possibly the neurological phenomenon termed the aura of migraine. It has been suggested that the type A stressful personality, often seen in the old characteristic descriptions of migraine sufferers, is associated with a Mg deficit..sup.48 The fact that most Americans exhibit shortfalls in Mg intake in their daily diets,.sup.34,35 and that some normal healthy volunteers exhibit deficits in serum IMg.sup.2+ but not TMg,.sup.49 supports the notion the Mg deficiency could be an important factor in the etiology of headaches.
Mg seems to possess unique weak calcium channel-blocking abilities..sup.5,35,36 It can act to regulate the entry of Ca.sup.2+ into smooth muscle and endothelial cells,.sup.35,36,50,51 and regulate the intracellular release and binding of Ca.sup.2+ 35,40,51 by acting on potential-operated, receptor-operated and leak-operated Ca.sup.2+ channels in these cells..sup.36 This may also help to explain why calcium channel blockers (CCB), unlike Mg.sup.2+ shown here, require a relatively long period of therapy (e.g., two-to-five months) to achieve reasonable therapeutic effects in migraine sufferers..sup.52 CCB could not be expected to act on leak-operated Ca.sup.2+ channels and only certain ones act on receptor-operated Ca.sup.2+ channel, making Mg.sup.2+ a potentially ideal therapeutic agent for headaches..sup.31
.sup.31 P-nuclear magnetic resonance spectroscopy showed low brain Mg.sup.2+ in a limited number of subjects during and between migraine attacks..sup.6 A low Mg.sup.2+ level is known to facilitate the development of spreading depression of Leao in animal experiments..sup.7 This phenomenon is thought to underlie the phenomenon of migraine aura. Several anecdotal observations and one double-blind, placebo controlled trial suggested that oral Mg supplementation may be helpful in some headache patients. Oral magnesium supplementation trials, for the treatment of headaches,.sup.9-12 may have been impeded in the past by two factors. Absorption of oral supplements is highly variable and IMg.sup.2+ levels were not monitored. One study reported that total serum Mg (TMg) levels were decreased in patients with migraine and tension-type headaches during an attack more than in the interictal period..sup.13 Two other studies showed normal serum levels of TMg between attacks of migraine..sup.12, 14 In one of these studies, a decreased TMg concentration in erythrocytes was found,.sup.14 while in the other a decreased TMg concentration in lymphocytes and polymorphonuclear cells, but not erythrocytes, was detected. A recent study showed decreased TMg content in mononuclear cells in migraine patients with and without aura during an interictal period..sup.15
Studies of IMg.sup.2+ in large numbers of headache patients showed different incidences of low IMg.sup.2+ levels in patients with different headache syndromes. Patients with chronic tension headaches.sup.19 had the lowest incidence (4.5%), while patients with episodic cluster headaches had a 50% incidence..sup.20 Patients with an acute migraine headache had a 42% incidence of low IMg.sup.2+ levels. Total magnesium levels in most of these patients, including those with low serum IMg.sup.2+, were normal..sup.19-22
The present invention is a novel treatment and composition that results in rapid and immediate relief of various types of headaches using magnesium.
A novel method to draw, handle, process and store biological samples for accurate, rapid and reproducible levels of ionized or free Mg.sup.2+ was developed. The method of collecting and processing samples has utility in preparing biological samples for measurement of ionized Mg.sup.2+ concentrations using a novel selective ion electrode with neutral carrier based membrane. Using the methods of the present invention, an accurate normal range for ionized Mg.sup.2+ in whole blood, plasma and serum has been determined for the first time. It is now possible to diagnose, prognoses and treat various disease states by the method of the present invention, by monitoring fluctuations in ionized Mg.sup.2+ concentrations.